The present invention relates to novel PON polypeptides and polynucleotides encoding same and to compositions and methods utilizing same. More particularly, the present invention relates to compositions including mutated PON polynucleotides or polypeptides and to methods of utilizing same for detoxification and decontamination, and for treating PON-associated diseases such as atherosclerosis.
Serum paraoxonases (PON1s) are calcium dependent phosphotriesterases which are essential to the detoxification process of organophosphates (OPs) such as the insecticide paraoxon and the nerve agents sarin and soman (Davies et al., 1996). Approximately 16% of the population is deficient in this enzyme and at high risk for damage from exposure to these and other OP agents, which are effected by the enzyme. PON1s also catalyze the hydrolysis of a broad range of carboxy-esters including lactones and thiolactones (Billecke et al., 2000; Jakubowski, 2000). PON1 resides within the cholesterol-carrying particles HDL (“good cholesterol”) and exhibits a multitude of activities related to the metabolism of drugs, lipids and other molecules associated with atheroscleorotic vascular and cardiac diseases (Ahmed et al., 2001; Billecke et al., 2000; Rodrigo et al., 2001). The levels of PON1 in the blood and its catalytic proficiency appear to have a major impact on susceptibility to athreosclerosis, cardiac and vascular diseases, cholesterol reducing drugs and various toxins and pollutants including insecticides (Smolen et al., 1991). It was also shown that mice lacking the PON1 gene are susceptible to atherosclerosis and organophosphate toxicity much more than PON1-carrying mice (Shih et al., 1998).
Despite its physiological and therapeutic importance, the structure and mechanism of action of PON1 have yet to be elucidated. PON1 appears to exhibit a curiously broad range of hydrolytic activities—catalyzing both phosphotriesters and carboxy-esters, as well as thiolactones (Billeclke et al., 2000). In addition, PON1 has been implicated in the reduction of lipid peroxides suggesting that it may also function as a peroxireductase (Aviram et al., 1998). Whether the latter is related to PON1's hydrolytic activities or not, is yet to be determined.
Almost all the research on PON1 has been performed on protein samples purified from sera. However, the yields of sera-purified PON1 are low, and the intimate association of PON1 with HDL can result in contamination by other HDL-associated enzymes including PON3 (Ahmed et al., 2001). The newly-reported activities of PON1 are orders of magnitude lower than with its well-characterized substrates—For example, hydrolysis of homocysteine thiolactone by such purified enzymes is 2,800-25000 times lower than with phenylacetate depending on the enzyme preparation (Billeclke et al., 2000; Jakubowski, 2000). Such low activities may result from miniscule amounts of a contaminating enzyme, such as PON3 [i.e., a variant of PON1 which is also found in HDL; (Ahmed et al., 2001)], or other serum enzymes. The difficulties in characterizing PON1 are highlighted by the recent discussion regarding its hydrolytic activity with PAF [i.e., platelet activating factor; (Rodrigo et al., 2001)]. Whilst one set of experiments suggests that PAF hydrolysis is mediated by PON1 and is not due to contamination of the purified PON1 with PAFAH (PAF acetyl hydrolase), a more recent publication argues that this activity is due to very low PAFAH contaminations (Marathe et al., 2002).
Amongst the issues yet to be clarified is the role of PON1 glycosylation in the enzyme's activity. Josse et al mutated two putative N-linked glycosylation sites (N252 and N323) of hPON1 expressed in human embryonic kidney cell line with no effect on its esterolytic activity (Josse et al., 1999). In contrast, enzymatic deglycosilation of hPON1 expressed in baculovirus abolished the enzyme's arylesterase activity, suggesting that glycosylation is essential to the enzyme's activity, and that sites other than N252 and N323 may be involved (Brushia et al., 2001). It was also shown that PON1 could not be functionally expressed in E. coli, presumably due to the absence of glycosylation in prokaryotes and the aggregation of the over-expressed protein into inclusion bodies (Brushia et al., 2001; Josse et al., 2002). PON1s also posses a disulphide bond, the formation of which may be hindered by the reducing environment present in E. coli's cytoplasm. Indeed, attempts to express hPON1 in E. coli under a broad range of conditions failed to yield soluble and active protein.
The availability of bacterially over-expressed PON1 that is soluble and catalytically active is of prime clinical value. Furthermore, the expression and purification of PON1 from E. coli can shed light on the different activities attributed to the enzyme, making it possible to explore its less pronounced activities while unambiguously ruling out contamination by other mammalian enzymes. The mechanism of PON1 activity could be investigated using biophysical methods which require high amounts of purified protein. Finally, functional expression in E. coli is the key for future attempts to engineer or directly evolve PON1 to have improved catalytic efficiencies towards therapeutic targets such as highly toxic nerve agents, or cardiac and vascular diseases related substrates such as lipid peroxides and homocysteine thiolactone.
Low solubility of proteins expressed in host systems is a major obstacle in the structural and functional characterization of numerous proteins (Waldo, 2003). Several methods have been developed to screen for mutant proteins with increased solubility. One approach using GFP fusion protein as a folding reporter is based on the correlation between folding of the target protein and the fluorescence of the E. coli cells expressing the GFP fusion (Waldo et al., 1999; Yang et al., 2003). Another approach is based on fusion of chloramphenicol acyltransferase (CAT) to the target protein allowing soluble mutants to be selected by growths at high levels on chloramphenicol (Maxwell et al., 1999). The main drawback of these approaches is, that selection pressure for solubility can only generate soluble mutant proteins with significant structural alterations and no function. In contrast, a direct screening for function ensures that soluble protein variants retain function [for the evolution of a soluble galactose oxidase in E. coli by a functional screen see Sun et al. (2001)].
While reducing the present invention to practice, the present inventors employed a directed evolution approach in order to engineer highly expressed recombinant variants of PON exhibiting activity spectra comparable to that of respective wild-type PONs purified from sera. Using the same approach, the present inventors also generated PON variants with specialized catalytic activities.
As is further described in the Examples section which follows, the availability of recombinant PON variants exhibiting kinetic parameters similar to those reported for PONs purified from sera enabled elucidation of the three dimensional (3D) structure of PON1, shedding light on its unique active site and catalytic mechanism. These findings allow, for the first time, to generate and use PON enzymes with improved catalytic efficiencies towards therapeutic targets such as highly toxic nerve agents, or cardiac and vascular diseases related substrates such as lipid peroxides and homocysteine thiolactone.